authors - university of stirling
TRANSCRIPT
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Title 1
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The effects of combined phytogenics on growth and nutritional physiology of Nile tilapia 3
Oreochromis niloticus 4
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Authors 6
Margaret Aanyu1,2
, Mónica B. Betancor1, Óscar Monroig
3* 7
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Addresses 9
1 Institute of Aquaculture, Faculty of Natural Sciences, University of Stirling, Stirling FK9 10
4LA, Scotland, United Kingdom 11
2 National Fisheries Resources Research Institute, Aquaculture Research and Development 12
Center, P. O. Box 530, Kampala, Uganda 13
3 Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Ribera de Cabanes 12595, 14
Castellón, Spain 15
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*Corresponding author17
Óscar Monroig 18
Tel: +34 964 319500; Fax: +34 964 319509; E-mail: [email protected] 19
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Accepted refereed manuscript of: Aanyu M, Betancor M & Monroig O (2020) The effects of combined phytogenics on growth and nutritional physiology of Nile tilapia Oreochromis niloticus. Aquaculture, 519, Art. No.: 734867. DOI: https://doi.org/10.1016/j.aquaculture.2019.734867.© 2019, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/
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Abstract 23
This study investigated whether dietary supplementation of phytogenic compounds 24
limonene and thymol had synergistic or additive effects on growth and selected nutritional 25
physiology pathways in Nile tilapia. A 63-day feeding experiment was conducted using 26
fish of 1.5 ± 0.0 g (± standard error) fed on a commercial diet coated with either 0 ppm 27
limonene and thymol (control), 400 ppm limonene (L), 500 ppm thymol, (T) or a 28
combination of 400 ppm limonene and 500 ppm thymol (LT). Final fish weight (FW) was 29
significantly improved to similar extents by diet LT (16.7 ± 0.3 g) and L (16.6 ± 0.4 g). 30
Dietary thymol alone and the control did not enhance FW (15.0 ± 0.4 g and 13.7 ± 0.4 g 31
respectively). Dietary thymol had shown a strong tendency to improve somatic growth 32
(P=0.052). The analysed candidate genes involved in the pathways of nutrient digestion, 33
absorption and transport (muc), lipid metabolism (lpl), antioxidant enzymes (cat) and 34
somatotropic axis growth (igf-I) were also up-regulated to similar extents in Nile tilapia by 35
diet L and LT (P<0.05), above the regulation observed with the diet supplemented 36
exclusively with thymol. This suggests lack of synergistic or additive effects on growth 37
and nutritional physiology pathways when limonene and thymol are supplied in the diet. 38
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Keywords 40
Growth promoters, limonene, Nile tilapia, nutritional physiology, phytogenic compounds, 41
thymol 42
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1. Introduction 44
Phytogenic compounds are natural bioactive compounds derived from herbs, shrubs and 45
spices with essential oils extracted from the plant parts being the major source of 46
phytogenic compounds (Yang et al., 2015; Sutili et al., 2017; Upadhaya and Kim, 2017). 47
Each phytogenic compound contains several bioactive components or molecules in 48
different proportions with bioactive components present in higher proportions largely 49
determining the biological properties of the essential oils (Santos et al., 2011; Chakraborty 50
et al., 2014; Yitbarek, 2015). Bioactive components in the plants are mainly hydrocarbons 51
(e.g. terpenes, sesquiterpenes), oxygenated compounds (e.g. alcohol, aldehydes, ketones) 52
and a small percentage of non-volatile residues (e.g. paraffin, wax) (Losa, 2000). Some 53
active compounds such as thymol, carvacrol, limonene, cinnamaldehyde and eugenol from 54
the plants thyme, oregano, citrus, cinnamon and clove respectively have been noted to 55
exert positive effects on nutrition, performance or health of monogastric animals (Wallace 56
et al., 2010; Chakraborty et al., 2014; Sutili et al., 2017; Upadhaya and Kim, 2017). 57
When mixtures of phytogenic compounds are used in animal feed, they can either have 58
synergistic, additive, indifferent or antagonistic effects on growth and other response 59
indicators in monogastric animals (Bassole and Juliani, 2012; Costa et al., 2013; Abd El-60
hack et al., 2016; Valenzuela-Grijalva et al., 2017; Amer et al., 2018; Youssefi et al., 2019). 61
Synergistic or additive effects of phytogenic compounds on growth performance lead to 62
enhanced growth of animals above the levels attained when the compounds are supplied 63
individually (Windisch et al., 2008; Yang et al., 2015). In fish, the combination of thymol 64
and carvacrol is arguably the most investigated blend of phytogenic compounds for its 65
beneficial effects on growth (Zheng et al., 2009; Ahmadifar et al., 2011; Hyldgaard et al., 66
2012; Chakraborty et al., 2013; Ahmadifar et al., 2014; Peterson et al., 2014; Perez-67
Sanchez et al., 2015). For instance, Peterson et al. (2014) reported that channel catfish 68
(Ictalurus punctatus) fed on a diet supplemented with a combination of limonene, thymol, 69
carvacrol and anethol gained 44% more weight than the control attributed to synergistic or 70
additive effects of the phytogenic compounds. 71
Often though indifferent effects can be observed when combination of phytogenic 72
compounds show no differences compared to treatments consisting of their individually 73
supplied compounds or the controls (Bassole and Juliani, 2012). Indifferent effects have 74
also been noted when combination of phytogenic compounds exerts significantly higher 75
effects to similar extents with only some treatments composed of their individually 76
supplied phytogenic compounds. Zheng et al. (2009) supplemented diets with 500 ppm of 77
either carvacrol, thymol or a mixture of carvacrol and thymol, and found a significantly 78
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higher weight gain of channel catfish with the dietary mixture of carvacrol and thymol 79
compared to the control and diet with thymol alone, but not with the diet supplemented 80
only with carvacrol. In addition, antagonistic effects occur when individual phytogenic 81
compounds might have positive effects but their combination results in negative effects 82
compared to the controls (Bassole and Juliani, 2012). Such antagonistic effects derived 83
from phytogenic blends have been often attributed to high concentrations of these 84
compounds that potentially result in unpleasant taste and smell and thereby retarding feed 85
intake and consequently growth (Windisch et al., 2008; Steiner, 2009; Costa et al., 2013; 86
Colombo et al., 2014). 87
The different responses to phytogenics mentioned above highlights the importance of 88
identifying combinations and doses of phytogenic compounds resulting in additive and 89
synergistic effects on fish growth. In our previous study (Aanyu et al., 2018), two 90
phytogenic compounds, namely limonene and thymol, classified as monoterpene and 91
diterpene, respectively, were found to have growth-promoting tendencies in Nile tilapia 92
(Oreochromis niloticus). We hypothesised that combinations of limonene and thymol can 93
potentially have additive or synergistic effects on the growth of Nile tilapia. Consequently, 94
this study aimed to investigate the effects of a blend of limonene and thymol, compared 95
with each of the compounds individually, on the growth, feed efficiency and nutritional 96
physiology of Nile tilapia. The study followed a candidate gene approach to investigate 97
physiological pathways underpinning the response of fish to the phytogenic compounds. A 98
selection of marker genes within the pathways of somatotropic axis-mediated growth, 99
nutrient absorption and transport, lipid metabolism and antioxidant enzyme status that 100
showed potential to be regulated by limonene and/or thymol were analysed (Aanyu et al., 101
2018). 102
103
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2. Materials and Methods 105
2.1 Ethical statement 106
All experiments were subjected to ethical review and approved by the University of 107
Stirling through the Animal and Welfare Ethical Review Body. The project was conducted 108
under the UK Home Office in accordance with the amended Animals Scientific Procedures 109
Act implementing EU Directive 2010/63. 110
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2.2 Experimental design 112
The feeding trial was carried out at the Aquaculture Research and Development Center 113
(ARDC), Uganda between March and May 2015. Nile tilapia juveniles from the same 114
cohort were obtained from the ARDC fish farm, acclimatized and size graded to 1.54 ± 0.0 115
g (mean ± standard error). Thirty eight (38) fish were stocked in each of the 16 116
experimental tanks. Each tank had a water holding capacity of 60 L, in a flow through 117
system with a flow rate of 1-2 L min-1
. The water in each tank was aerated using air stones 118
and heated using aquaria water heaters to 25.0 - 26.6 oC. A photoperiod of 12h light-12h 119
dark was maintained. 120
Water quality was monitored routinely to ensure that it was within the requirements 121
for Nile tilapia growth (Lim and Webster, 2006). A multi-parameter meter (HQ40D model, 122
Hach Ltd Germany) was used to measure dissolved oxygen, pH and water temperature. 123
The level of ammonia-nitrogen was assessed using a fresh water test kit from API 124
Company Ltd UK following the user guide from the manufacturer. Water flowing into the 125
fish rearing tanks had 6.6 ± 0.6 mg L-1
of dissolved oxygen, pH 6.8 ± 0.3, undetectable 126
levels (< 0.05 mg L-1
) of ammonia-nitrogen and water temperature ranging from 23.3 - 127
24.3 oC before heating with aquaria water heaters. 128
129
2.3 Experimental diets 130
A standard commercial feed (CP35%, Kampala, Uganda) for juvenile Nile tilapia 131
produced at the ARDC was supplemented with limonene (97 % purity) and/or thymol 132
(95 % purity) from Sigma Aldrich, Kampala, Uganda using concentrations found to have 133
growth-promoting potential in Nile tilapia (Aanyu et al., 2018). The diets included: 0 ppm 134
limonene and thymol (Control); 400 ppm limonene (L); 500 ppm thymol (T); and a 135
combination of 400 ppm limonene and 500 ppm thymol (LT). In order to supply the above 136
concentrations of phytogenic compounds to the feed, each concentration of phytogenic 137
compounds was prepared in 100 mL of absolute ethanol and sprayed onto 1 kg of feed. 138
The control was also coated with a similar amount of ethanol but no phytogenic compound 139
was added. All diets were air-dried for one day, packed in airtight polythene bags and 140
stored at room temperature until use. 141
Each experimental diet was tested in quadruplicate tanks and the treatments were 142
distributed randomly. The fish were fed the experimental diets twice a day to apparent 143
satiation and the feed intake was recorded. 144
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The proximate nutritional composition (dry matter, moisture, protein, lipid, fibre, ash 145
and gross energy) of the standard diet was determined according to the methods of the 146
Association of Official Analytical Chemists (AOAC, 1990) and the joint technical 147
committee of the International Organisation for Standardisation and International 148
Electrotechnical Commission (ISO/IEC 17025). Briefly, dry matter content was estimated 149
by drying a sample of feed in an oven at 105-110˚C to a constant weight and the 150
percentage retained weight from the original sample was the amount of dry matter whereas 151
the percentage loss in weight of the sample was the moisture content. Crude protein 152
content was determined using the Kjeldahl method and crude lipid by petroleum ether 153
extraction using the Soxhlet method. Crude fibre content was analysed by acid / alkaline 154
hydrolysis of a sample and the amount of insoluble residues resistant to hydrolysis was the 155
fibre content. Crude ash was determined by combustion of a sample in a furnace at 600 ˚C 156
for 24 h. The gross energy using determined using the bomb calorimetry method. 157
Proximate composition of the standard diet used in this study is shown in Table 1. 158
159
2.3 Fish measurements and sample collection 160
Growth of the fish was estimated by measuring the weight (accuracy of 0.1 g) and total 161
length (0.1 cm) of all fish in each tank every three weeks and at the end of the experiment 162
(63 days). This procedure was carried out while fish were anesthetised using 0.02 g L-1
of 163
clove oil for 3-5 min, after which the fish were placed in aerated water and taken back to 164
the experimental tanks. At the end of the experiment, the number of live fish in each tank 165
was recorded for survival estimation, and sections of liver and fore intestine were dissected 166
from 16 fish per treatment (n=4 per tank) and placed in 1.5 mL tubes containing 167
RNAlater®
(Sigma Aldrich, Kampala, Uganda). The liver and fore intestine samples were 168
kept at 4 ºC overnight, shipped to the UK and transferred to a -20 oC freezer until RNA 169
was extracted. 170
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2.4 Fish performance calculations 172
The effects of the experimental diets on fish performance were assessed by calculating the 173
performance indicators below using the following formulae: 174
175
Final average fish weight (FW, g) = total fish biomass at end of the trial (g) / number of 176
fish at end of the experiment; 177
Percentage (%) weight gain (WG, %) = ((final average fish weight (g) - initial average fish 178
weight (g)) / initial average fish weight (g)) × 100; 179
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Condition factor (CF) = (final average fish weight / final average total length3) × 100; 180
Fish survival rate (SR, %) = (number of alive fish at end of trial / initial number of fish 181
stocked) × 100 %; 182
Feed intake (FI, % body weight per day) = (100 × (average feed intake fish-1
/ ((initial 183
average body weight ± final average body weight)/2))) / duration of trial (d); 184
Feed conversion ratio (FCR) = average feed intake fish-1
/ weight gain; 185
Protein efficiency ratio (PER) = weight gain / protein intake. 186
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2.5 Molecular analyses 188
2.5.1 RNA extraction and complementary DNA (cDNA) synthesis 189
Tissue samples from the liver and fore intestine were homogenised in TRI Reagent (Sigma 190
Aldrich, Dorset, UK) with a mini bead-beater 16 (Biospec Bartlesville, OK, USA), and 191
total RNA was extracted from the samples (n=16 per tissue and treatment). The 192
concentration and purity of the RNA was measured by spectrophotometry with an ND-193
1000 Nanodrop (Nanodrop 1000, Thermo Scientific, Glasgow, UK). The integrity of the 194
RNA (aliquots fo 200 ng total RNA) from each sample was further assessed by agarose gel 195
(1 %, v/v) electrophoresis. 196
From a total of 16 RNA samples extracted per tissue and treatment, eight RNA samples 197
were derived by pooling together two samples from the same tank and treatment, taking 198
equal quantity (2.5 µg µl-1
) of RNA from each of the two samples being pooled (adapted 199
from Glencross et al., 2015). Thus the final mixture (vortex mixed and centrifuged) had a 200
50 % contribution of each of the two samples that were pooled. 201
A high capacity reverse transcription kit without RNase inhibitor from AB Applied 202
Biosystems (Warrington, UK) was used to reverse transcribe 2 µg µl-1
RNA from each 203
pool sample (n = 8 per treatment) to cDNA following the protocol provided by the 204
manufacturer. 205
206
2.5.2 Quantitative real-time Polymerase Chain Reaction (qPCR) 207
The mRNA expression levels of selected genes of interest in the pathways of somatotropic 208
axis-mediated growth, nutrient absorption and transport, lipid metabolism and antioxidant 209
enzyme status were analysed by quantitative real-time polymerase chain reaction (qPCR) 210
in tissue samples (liver or fore intestine) in which they perform their major biological 211
functions. The selected target genes included mucin-like protein (muc), oligo-peptide 212
transporter 1 (pept1), lipoprotein lipase (lpl), sterol regulatory element binding 213
transcription factor 1 (srebf1), alkaline phosphatise (alp), phospholipase A2 (pla2), 214
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catalase (cat), growth hormone (gh), and insulin growth factor I (igf-I). Efficiency of the 215
primers was first tested by generating standard dilution curves, assessing the melting 216
curves and cycle threshold (Ct) values (Larionov et al., 2005). Efficient primers were 217
considered to have values between 0.80 - 1.10, a single melting peak, Ct value below 30 218
and one clear band under 1 % agarose gel electrophoresis. The details of the primers used 219
for qPCR analyses are provided in Table 2. Each qPCR contained duplicate samples (total 220
volume 20 µl each) containing 5 µl of 20-fold diluted cDNA, 3 µl nuclease-free water, 1 μl 221
(10 pmol) each for the forward and reverse primers, and 10 μl of Luminaris color higreen 222
qPCR Mix (Thermo Scientific, Hemel Hempstead, UK). All the reactions were run in 96 223
well-plates using a Biometra TOptical Thermocycler (Analytik Jena, Goettingen, 224
Germany). A calibrator sample (20-fold dilution of all samples pooled cDNA) and a 225
negative control with no cDNA (non-template control-NTC) were included in each plate. 226
The qPCR thermocycling program involved pre-heating samples at 50 °C for 2 min 227
followed by 35 cycles, initial denaturing at 95 °C for 10 min, denaturation at 95 °C for 15 s, 228
annealing at 60 °C for 30 s, and an extension at 72 °C for 30 s. 229
230
2.5.3 Gene expression computations 231
Ct values for the duplicate runs of each sample were averaged. The data were normalised 232
using the geometric mean expression of the references genes (β-actin and ef-1α) and the 233
relative expression of each gene was calculated according to the equation of Pfaffl (2001). 234
Heat maps enabling cluster analysis and visualisation of the expression patterns of the 235
analysed genes were generated but not based on statistical differences. Java Tree View 236
Software was used to plot the data and perform cluster analysis based on Euclidean 237
distance. Expression level of each gene was natural log transformed and normalised 238
against two reference genes (β-actin and ef-1α). 239
240
2.6 Statistical analysis 241
The data were analysed using the Statistical Package for the Social Sciences (SPSS) 242
version 19 (Chicago, USA) (Landau and Everitt, 2004). The fish performance and qPCR 243
results are expressed as means ± standard error. Normality of distribution of the data was 244
assessed using Kolmogorov-Smirnov’s tests. Data not normally distributed were subjected 245
to natural logarithm ln (qPCR data) and arcsin square-root (WG, CF, SR, FI, FCR, and 246
PER) transformation. Differences among dietary treatments were analysed by one-way 247
ANOVA followed by Tukey’s test. When heterogeneity of variances occurred, Welch's test 248
was performed with Game-Howell's test to determine differences between treatments. 249
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Significant differences were considered at P value < 0.05. In addition, Pearson’s 250
correlation analysis was performed to indicate the relationship and degree of correlation 251
between FI and FCR, FW and SR, FW and PER. The significance level of correlation was 252
set to P < 0.05. 253
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3. Results 255
3.1 Fish performance 256
Table 3 shows the performance of Nile tilapia fed on diets supplemented with 400 ppm 257
limonene (L), 500 ppm thymol (T) and the combination of 400 ppm limonene with 500 258
ppm thymol (LT) for 63 days (9 weeks). There was a significant increase (P < 0.01) in the 259
final weight of fish fed on the diets supplemented with limonene, that is, diets L and LT, 260
compared to the control. No significant increases in fish weight were observed with diet L 261
and LT during day 1, 21, and 42 of the feeding experiment (data not shown). The diet 262
supplemented exclusively with thymol (T) did not significantly improve the final weight of 263
the fish (P = 0.052). There was a significantly higher percentage weight gain (% WG; P = 264
0.01) of fish fed on diets L and LT compared to the control, whilst fish fed diet T did not 265
show significant differences compared with the control. Despite there were no significant 266
differences in the final survival of the fish among treatments, there was a strong significant 267
positive correlation (r = 0.967, P = 0.033) between the survival rate and final weight of the 268
fish (Table 3). Condition factor (CF) was not significantly different among treatments. 269
The protein efficiency ratio (PER) was significantly higher in fish fed the diets L and LT 270
compared to the control. While no significant differences in PER were observed among 271
fish fed on diets L, T and LT, PER had a strong significant positive correlation (r = 0.974, 272
P = 0.026) with final fish weight and thus higher PER corresponded with higher final fish 273
weight. This study found no significant differences in feed conversion ratio (FCR) among 274
the treatments supplemented with L, T and LT, but fish fed diets L and LT had 275
significantly lower FCR (P = 0.006) than the control-fed fish (Table 3). Despite % feed 276
intake (% FI) not being significantly different among fish fed diets L, T and LT, 277
significantly lower (P = 0.019) FI was obtained with the fish fed on diets L and LT 278
compared with the control. In addition, there was a strong positive significant correlation (r 279
= 0.996, P = 0.004) between FI and FCR, and lower FI corresponded with low FCR and 280
therefore better feed utilisation efficiency. 281
282
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3.2 Relative mRNA gene expression 283
The heat map in Figure 1 represents the relative expression patterns (not based on 284
statistical differences) of genes analysed in the liver (a) and fore intestine (b) of Nile tilapia 285
fed on the experimental feeds. There were more genes with patterns of higher relative 286
expression levels (red) among the fish fed on diets L and LT compared with diet T, when 287
all dietary treatments were compared to the control. 288
289
3.2.1 Expression of genes involved in somatotropic axis in liver 290
Insulin growth factor I (igf-I) was significantly (P = 0.025) up-regulated in the liver of fish 291
fed on diets L and LT compared with control fish (Figure 2). However, the expression of 292
igf-I did not differ significantly between the fish fed on diets L, T and LT (Figure 2). In 293
addition, the relative expression levels of gh was not significantly different in the livers of 294
fish fed on diets L, T, LT and the control. 295
296
3.2.2 Expression of genes involved in lipid metabolism in liver 297
The expression of lpl, alp and srebf1 in the liver of Nile tilapia fed on the experimental 298
diets is shown in Figure 3. Levels of lpl mRNA were significantly (P = 0.003) higher in 299
fish fed on diet LT compared with the control. The expression of lpl in the fish fed on diets 300
L and T was not significantly different from the control. Similarly, no significant 301
differences in the relative expression of alp and srebf1 were found among the experimental 302
treatments (Figure 3). 303
304
3.2.3 Expression of genes regulating nutrient digestion, absorption and transport in the fore 305
intestine 306
The mRNA levels of muc were significantly higher (P = 0.025) in the fore intestine of fish 307
fed on diet LT compared with the control (Figure 4). Besides, the expression of muc in the 308
fish fed on diets L and T did not differ significantly from the control (P = 0.097). The 309
expression of pla2 also did not statistically differ among the dietary treatments (P = 0.086). 310
Oligo-peptide transporter 1 (pept1) expression was significantly (P = 0.047) up-regulated 311
in the fish fed on diet LT compared with the control, although expression levels in fish fed 312
diets L and T did not differ statistically compared with the control (Figure 4). 313
314
3.2.4 Expression of antioxidant enzymes in liver 315
The expression of cat was significantly higher (P = 0.006) in the liver of fish fed on diets L 316
and LT compared with the control (Figure 5). 317
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4. Discussion 320
The present study investigated the effects of diets containing limonene and thymol, 321
supplemented both individually and in combination, on growth and nutritional physiology 322
of Nile tilapia. The goal was to establish whether blends of limonene and thymol had 323
synergistic and/or additive effects on the growth of Nile tilapia. A selection of gene 324
markers regulating nutrient digestion, absorption and transport, lipid metabolism, 325
antioxidant enzymes and somatotropic axis growth were investigated. 326
Fish fed diets supplemented with limonene alone (L) and the blend of limonene and 327
thymol (LT) had significantly higher FW, % WG, PER and lower FI and FCR than the 328
control. On the contrary, fish fed on the diet supplemented with only thymol (T) did not 329
show any statistical difference in such parameters compared with the control. Similarly, 330
Zheng et al. (2009) found no synergistic or additive effects of a combination of carvacrol 331
and thymol on the weight gain of channel catfish. The fish fed on the diet supplemented 332
with only carvacrol, and the blend of carvacrol and thymol attained statistically higher 333
weight gain compared with the diet with only thymol and the control, but the dietary 334
mixture of carvacrol and thymol did statistically increase weight gain to the same extent as 335
the diet with only carvacrol. 336
Among the feed utilisation parameters, the present study found enhanced feed 337
efficiency (i.e., lower FCR) in fish fed diets L and LT, and a strong significant positive 338
correlation between PER and FW. The correlation between PER and FW showed that, as 339
the utilisation of protein from the feed was enhanced (high PER), FW of the fish was 340
increased. This could have contributed to the significantly improved somatic growth of the 341
fish fed on diets L and LT, both treatments with increased PER compared to the control. 342
The increased WG and lowered FI levels observed with diet L and LT fed fish are in 343
agreement with Hashemipour et al. (2013) who found lower FI corresponding with the 344
highest WG and feed efficiency in broiler chicken fed on a diet with a mixture of 200 ppm 345
of thymol and carvacrol compared to the control. It is known that efficient growth in fish 346
does not necessarily coincide with maximum or higher FI because fish adjust their FI 347
according to their energy requirements (Ali and Jauncey, 2004), with better feed efficiency 348
occurring below maximum FI (Rad et al., 2003; Sawhney, 2014). Conversely, some studies 349
with phytogenic compounds (thymol and carvacrol) in pig diets found low FI 350
corresponding with low WG (Lee et al., 2003a; Lee et al., 2003b; Zhai et al., 2018). While 351
it is difficult to identify the exact causes of such an apparent discrepancy with the present 352
results, one possible reason might stem from the pungent odour of thymol and carvacrol 353
13
that can affect palatability and ultimately feed intake since, compared to fish, pigs are more 354
sensitive to smell (Michiels et al., 2012; Muthusamy and Sankar, 2015). 355
The actions of genes regulating growth in the pathways within nutritional physiology are 356
complementary to each other (Hashemi and Davoodi, 2010; Steiner and Syed, 2015). In 357
this study, insulin growth factor I (igf-I), which plays a core role in regulating growth in 358
the somatotropic axis, was up-regulated to a similar extent in the liver of fish fed diets L 359
and LT, corresponding also to higher final FW and feed utilisation efficiency (FCR) than 360
the control. This observation implies that igf-I was largely activated by limonene 361
suggesting that there was no synergistic or additive effect of limonene and thymol in 362
influencing somatotropic axis-mediated growth. 363
Key mechanisms underlying feed utilisation efficiency include nutrient digestion, 364
absorption and transport, in which mucin-like protein (muc) and oligo-peptide transporter 1 365
(pept1) are important components (Verri et al., 2011; Fascina et al., 2012). The present 366
study found a significantly higher expression of muc in the fore intestine of fish fed on diet 367
LT compared with the control, with diets L and T showing no differences in expression of 368
muc with the control and diet LT. The high expression of muc found with diet LT can be 369
associated with an increase in the secretion/quantity of mucus, which then serves as a 370
lubricant aiding absorption of nutrients into the bloodstream through which they are 371
transferred to tissues for various functions including growth (Kamali et al., 2014). 372
Moreover, high expression of muc corresponded with enhanced somatic growth of the fish 373
in the LT treatment. Despite that Tsirtsikos et al. (2012) did not specifically investigate 374
muc expression, their study on broilers fed on diets containing a blend of limonene, 375
carvacrol and anethol also reported an increase in mucus volume in the fore intestine. 376
Additionally, Jamroz et al. (2006) found higher mucus secretion in the fore intestine of 377
broilers fed diets supplemented with a combination of phytogenics including carvacrol, 378
cinnamaldehyde and capsicum oleoresin. The present results for Nile tilapia are consistent 379
with these terrestrial animal studies, suggesting that the mechanism of action is somewhat 380
conserved across vertebrates. 381
The movement of nutrients from the lumen of the intestine, aided by mucus, into 382
epithelial cells takes place through diffusion and/or active transport regulated by nutrient 383
transporters (Rust, 2003). The nutrient transporter pept1 that aids the transport of protein in 384
the form of di/tri peptides through the above process (Verri et al., 2011), was significantly 385
regulated by diet LT compared with the control. Moreover, the higher expression of pept1 386
in fish fed diet LT corresponded with significantly improved feed efficiency (lower FCR) 387
and PER compared with the control, with diet L also having enhancing feed efficiency 388
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(lower FCR) and PER compared with the control. This suggested that limonene drove the 389
improved protein absorption, which could have contributed to increased growth. Similarly, 390
dietary peppermint and Digestarom P.E.P (Biomin GmbH, Herzogenburg, Austria), a 391
commercial matrix-encapsulated phytogenic mixture, improved protein utilisation in 392
broilers (Upadhaya and Kim, 2017) and gilthead seabream Sparus aurata (Goncalves and 393
Santos, 2015). 394
In order to maximise the use of dietary protein for somatic growth, energy for 395
supporting metabolic processes can be derived from non-protein sources, particularly lipids 396
(Nankervis et al., 2000). Lipid metabolism including, among others, processes such as lipid 397
catabolism or fatty acid and triglyceride synthesis occurs along with lipid transport and 398
deposition with the liver as the main active site (He et al., 2015). In the present study, diet 399
LT activated lipid metabolism as reflected by significantly increased expression of 400
lipoprotein lipase (lpl) in comparison to the control. Since the expression of lpl in the fish 401
fed diet T did not differ from that of the control, it is reasonable to deduce that limonene, 402
not thymol, is the compound that triggers such metabolic response in fish fed diet LT. 403
Given that lpl plays a pivotal role in breaking down plasma lipids into free fatty acids and 404
transporting them for use in energy production (Tian et al., 2015), the high gene expression 405
of lpl found in this study suggests that dietary limonene increased the energy level of the 406
fish, thereby providing sufficient energy for running metabolic processes and sparing 407
protein, which significantly improved fish growth in the dietary treatments L and LT. Such 408
effect of limonene to regulate lpl and a corresponding somatic growth enhancement further 409
confirmed the results obtained by Aanyu et al. (2018) in the same teleost species. 410
Metabolic processes in the body result into production of reactive oxygen intermediates 411
(ROIs), which can induce damage to cells and tissues if their levels are not maintained low 412
(Covarrubias et al., 2008; Costa et al., 2013) This can ultimately impair adequate 413
physiological function and subsequently negatively affect growth. In this study, the 414
expression of catalase (cat), a key antioxidant enzyme that breaks down the ROI hydrogen 415
peroxide, was significantly increased by dietary treatment with limonene (i.e., treatments L 416
and LT) to similar extents. These results suggest that the enhanced antioxidant status by 417
cat could reduce the hydrogen peroxide levels and thus result in improved somatic growth 418
of the fish fed on diet L and LT. Recent research has shown that, when ROIs are at low 419
concentrations, they are vital molecules mediating physiological processes including 420
somatic growth (Covarrubias et al., 2008; Barbieri and Sestili, 2012). The herein reported 421
action of dietary limonene on cat up regulation (catalase enzyme activity) has not been 422
observed for other phytogenic compounds. For instance, Zheng et al. (2009) did not find 423
15
enhanced activity of catalase enzyme in channel catfish fed on diets containing thymol, 424
carvacrol or their mixture, although the fish attained higher weight with the diet containing 425
both compounds and carvacrol alone. Thymol did not appear to have an obvious role in 426
regulation of antioxidant enzymes such as cat, and thus it can be assumed that, as noted 427
above, limonene exerts a major action in up-regulating cat. 428
429
5. Conclusions 430
This study confirmed that dietary limonene and the blend of limonene and thymol 431
improved somatic growth and feed utilisation efficiency of Nile tilapia to similar extents, 432
although thymol individually showed no effects on enhancing growth performance. This 433
indicated that dietary limonene was the major contributor towards the enhanced fish 434
growth observed, suggesting lack of synergistic or additive effects of the combined 435
compounds. The gene expression of biomarkers for nutrient digestion, absorption and 436
transport, lipid metabolism, antioxidant enzymes and somatotropic axis growth also largely 437
showed lack of synergistic or additive effects of the dietary combination of limonene and 438
thymol in Nile tilapia. 439
440
Acknowledgements 441
This work was funded by the Schlumberger Foundation PhD Training Program, and the 442
Government of Uganda through the Agricultural Technology and Agribusiness Services 443
(ATAAS) Project of the National Agricultural Research Organization (NARO). 444
445
Data Availability Statement 446
The data that support the findings of this study are available from the corresponding author 447
upon reasonable request. 448
449
16
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21
590 FIGURES 591 592
FIGURE 1 Heat map showing the expression patterns of nine genes analysed using qPCR 593
data from Nile tilapia fed on diets supplemented with limonene (L), thymol (T) and their 594
combination (LT). Data were plotted using Java Tree View and rows were clustered 595
according to Euclidean distance. The columns represent mean data values of three dietary 596
treatments L (400 ppm limonene), T (500 ppm thymol) and LT (400 ppm limonene and 597
500 ppm thymol). The rows indicate each of the analysed genes in the liver (a) and fore 598
intestine (b) of Nile tilapia. Expression level of each gene was natural log transformed and 599
normalised against two reference genes. The colour bars at the bottom represent the mean 600
relative expression levels as low (green), neutral (black) or high (red). The black colour 601
represents the control to which the relative expression of the other treatments was 602
determined. cat, catalase; gh, growth hormone; srebf1, sterol regulatory element binding 603
transcription factor 1;alp, alkaline phosphatase; igf-I, insulin growth factor I; pla2, 604
phospholipase A2; lpl, lipoprotein lipase; muc, mucin-like protein; pept1, oligo-peptide 605
transporter 1. 606
607 608
609 610
L
cat
gh
srebf1
alp
igf-I
T LT
lpl pept1
muc
pla2
L T LT
(a) (b)
22
611
FIGURE 2 Expression of insulin growth factor I (igf-I) and growth hormone (gh) in the 612 liver of Nile tilapia fed on diets supplemented with 400 ppm limonene (L), 500 ppm 613 thymol (T) and the combination of 400 ppm limonene and 500 ppm thymol (LT). All 614 values are means of treatments ± standard error (n=8). Different superscript letters denote 615 significant differences in the expression of igf-I between the treatments. 616
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Control L T LT
Rel
ati
ve
gen
e ex
pre
ssio
n l
evel
Experimental diets
igf-1 gh
a
b ab
b
23
617
FIGURE 3 Expression of lipoprotein lipase (lpl), alkaline phosphatase (alp), and sterol 618
regulatory element binding transcription factor 1 (srebf1) in the liver of Nile tilapia fed on 619
diets supplemented with 400 ppm limonene (L), 500 ppm thymol (T) and a combination of 620
400 ppm limonene and 500 ppm thymol (LT). All values are means of treatments ± 621
standard error (n=8). Different superscript letters denote significant differences in the 622
expression of lpl between the treatments. 623
624
625
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Control L T LT
Rel
ati
ve
gen
e ex
pre
ssio
n l
evel
Experimental diets
lpl alp srebf1
a
ab
a
b
24
626
FIGURE 4 Expression of mucin-like protein (muc), phospholipase A2 (pla2), and oligo- 627
peptide transporter 1 (pept1) genes in the fore intestine of Nile tilapia fed on diets 628
supplemented with 400 ppm limonene (L), 500 ppm thymol (T) and a combination of 400 629
ppm limonene and 500 ppm thymol (LT). All values are means of treatments ± standard 630
error (n=8). For each gene (muc or pept1), different superscript letters denote significant 631
differences in the expression of each gene between the treatments. 632
633 634
0.0
0.5
1.0
1.5
2.0
2.5
Control L T LT
Experimental diets
Rel
ati
ve
gen
e ex
pre
ssio
n lev
el
muc
pla2
pept1
ab ab
b
a
ab
b
a
ab
25
635 FIGURE 5 Expression of the antioxidant enzyme catalase (cat) in the liver of Nile tilapia 636
fed on diets supplemented with 400 ppm limonene (L), 500 ppm thymol (T) and a 637
combination of 400 ppm limonene and 500 ppm thymol (LT). All values are means of 638
treatments ± standard error (n=8). Different superscript letters denote significant 639
differences in the expression of cat between the treatments. 640
641
642
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Control L T LT
Rel
ati
ve
cat
exp
ress
ion
lev
el
Experimental diets
a
b
a
b
26
TABLES 643
TABLE 1 Proximate analysis of the nutritional composition of the diet (CP35, ARDC - 644 Uganda) used in the present trial to feed Nile tilapia (Oreochromis niloticus) for 63 days. 645
Analysis Quantity
Dry matter (%) 89.1
Moisture (%) 10.9
Crude protein (%) 33.1
Crude fat (%) 3.3
Crude ash (%) 10.9
Crude fibre (%) 9.9
Gross energy (Kj g-1
) 16.9
646 647 648
27
TABLE 2 Details of the primers used for quantitative real-time PCR analyses.
Functional group Gene symbol Primer / oligonucleotide sequences (5’-
3’)
Size
(base pairs)
Accession number*
Nutrient digestion,
absorption and
transport
muc F: TGCCCAGGAGGTAGATATGC 101 XM_005466350
R: TACAGCATGAGCAGGAATGC
pept1 F: CAAAGCACTGGTGAAGGTCC 196 XM_013271589
R: CACTGCGTCAAACATGGTGA
Lipid metabolism lpl F: TGCTAATGTGATTGTGGTGGAC 217 NM_001279753
R: GCTGATTTTGTGGTTGGTAAGG
srebf1 F: TGCAGCAGAGAGACTGTATCCGA 102 XM_005457771
R: ACTGCCCTGAATGTGTTCAGACA
alp F: CTTGGAGATGGGATGGGTGT 200 XM_005469634
R: TTGGCCTTAACCCCGCATAG
pla2 F: CTCCAAACTCAAAGTGGGCC 177 XM_005451846
R: CCGAGCATCACCTTTTCTCG
Antioxidant activity cat F: TCCTGGAGCCTCAGCCAT 79 JF801726
R:
ACAGTTATCACACAGGTGCATCTTT
Somatotropic axis-
aided growth
gh F: TCGGTTGTGTGTTTGGGCGTCTC 90 XM_003442542
R: GTGCAGGTGCGTGACTCTGTTGA
igf-I F: GTCTGTGGAGAGCGAGGCTTT 70 NM_001279503
R: CACGTGACCGCCTTGCA
Reference genes ef-1α F: GCACGCTCTGCTGGCCTTT 250 NM_001279647
R: GCGCTCAATCTTCCATCCC
ß-actin F: TGGTGGGTATGGGTCAGAAAG 217 XM_003443127
R: CTGTTGGCTTTGGGGTTCA
muc, mucin-like protein; pept1, oligo-peptide transporter 1; lpl, lipoprotein lipase; srebf1, sterol regulatory element binding transcription factor 1; alp,
alkaline phosphatase, pla2 phospholipase A2; cat, catalase; gh, growth hormone; igf-I, insulin growth factor I; ef-1α, elongation factor 1α; ß-actin beta-
actin.
29
TABLE 3 Growth, feed utilisation efficiency and survival rate of Nile tilapia fed on
diets with 400 ppm limonene (L), 500 ppm thymol (T) and a combination of 400 ppm
limonene and 500 ppm thymol (LT) for 63 days.
Parameter Experimental diet
Control L T LT P value
Initial mean weight (g) 1.5 ± 0.0 1.6 ± 0.0 1.5 ± 0.0 1.6 ± 0.0 NS
Final mean weight (g) 13.7 ± 0.4a 16.6 ± 0.4
b 15.0 ± 0.4
a 16.7 ± 0.3
b 0.001
% WG 793.2 ± 29.1a 957.3 ± 51.9
b 887.0 ± 16.1
ab 980.0 ± 41.3
b 0.011
CF 1.8 ± 0.0 1.8 ± 0.0 1.9 ± 0.0 1.9 ± 0.0 NS
% Survival 94.1 ± 3.5 97.4 ± 1.5 94.8 ± 3.0 98.1 ± 0.7 NS
% FI (% body weight d-
1)
4.5 ± 0.1b 3.9 ± 0.1
a 4.3 ± 0.2
ab 4.0 ± 0.1
a 0.019
FCR 1.8 ± 0.1b 1.5 ± 0.0
a 1.7 ± 0.1
ab 1.5 ± 0.0
a 0.027
PER 1.7 ± 0.1a 2.0 ± 0.1
b 1.9 ± 0.1
ab 2.0 ± 0.1
b 0.009
All values are means of treatments ± standard error. Mean values with different
superscript in the same row are significantly different from each other at P < 0.05. NS,
refers to not significantly different values. For each treatment, n =152 for initial fish
weight, for final fish weight, n = number of alive fish at the end of the trial, and n = 4
for percentage of weight gain (% WG), condition factor (CF), survival rate (%
survival), food intake (% FI), feed conversion ratio (FCR) and protein efficiency ratio
(PER).